Identification and characterisation of viral bloody diarrhoea aetiology in puppies, presented to the Animal Health hospital, North-West University

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Identification and characterisation of viral

bloody diarrhoea aetiology in puppies,

presented to the Animal Health hospital,

North-West University

K Ntumba

Orcid.org 0000-0003-1601-5314

Dissertation submitted in fulfilment of the requirements for the

degree

Master of Science in Animal Health

at the North-West

University

Supervisor:

Prof. M Mwanza

Co-supervisor: Dr. L Ngoma

Graduation ceremony: April 2020

Student number: 27046710

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DECLARATION

I declare that the work presented in this dissertation entitled “Identification and characterisation of viral bloody diarrhoea aetiology in puppies, presented to the Animal Health hospital, North-West University” submitted for the degree of Master of Science in Animal Health is my own design work and does not belong to any other dissertation. Furthermore, I declare that it has not been previously submitted at this or any other universities.

Dr Kayamba Ntumba April 2020

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DEDICATION

I dedicate my research to:

JESUS CHRIST almighty for giving me this life and the opportunity to achieve this research through his blessing, goodness and infinite mercy promised me to the highest ranking of life, education and careers. Thank you very much for being my true father.

My beloved wife Esther Maracto NTUMBA for the unconditional love, care, attention, inspiration and indescribable encouragement with a blend of affection as my esteemed guide and major advisor during the entire process of research work for completing this dissertation, you will always be the best. I am extremely grateful. And to my daughters Priscylla M. NANDA, Ronewa Vision SANDANI, Kaela M. NTUMBA and two sons Asher M. NTUMBA, Almar K. NTUMBA for their prayers, love, affection, sacrifices and for accepting the absence of their father when they needed me. You were a source of inspiration and courage during my study. May JESUS CHRIST keep blessing and protecting you forever.

I would like to express my gratitude and dedicate this work to my late father Mulumba Ntumba for introducing the concept of studying hard at a very young age, thank you very much Dad for being my Hero. May your Soul Rest In Peace. To my lovely mother Anastasie Mwauka for the prayers, love, inspiration, sacrifice and blessings every step. I’m extremely blessed to have such wonderful parents. I will always be grateful and thankful to them and promise to make you proud.

To the Ntumba family for their prayers, love, sacrifice and support during the process of my research work. I’m grateful to you all.

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ACKNOWLEDGEMENTS

I’m privileged to express my gratitude to Professor Mwanza Mulunda for his guidance, suggestions, comprehensive planning, continuous support and encouragement, constructive criticism and generous help are greatly acknowledged. His interest, whole hearted co-operation on the topic; enthusiastic support on my efforts and meticulous correction were a source of inspiration to achieve the work and I consider myself fortunate to have a wonderful supervisor. I owe a huge amount of gratitude to my co-supervisor Dr. Lubanza Ngoma for his suggestions, guidance, and continuous encouragement, supportive advice and his generous help and knowledge in the laboratory work are acknowledged.

I intend to express my gratitude to Miss T. F. Tshinavhe Muofhe for her care, suggestion, affection, perseverance encouragement, help in the laboratory work and inexhaustible criticism step by step on my research. I am extremely blessed to have such a wonderful friend. I’m and will always be thankful and grateful.

I have a deep obligation to thank the team of North West University hospital: Ms T.P. Ateba, Mr. Thatho Moroane, Regiana Dichaba, Dr. Jullian, Dr. Bianca, and Fifi Refilwe for the wonderful help on the collection of samples. I am really grateful for your inestimable help. I would like also to acknowledge Professor Michelo Syakalima, Dr. R. Victress Ndou for the constant support, advice and encouragement at every step.

I take this unique opportunity to show my deepest sense of gratitude and gratefulness to the Department of higher education and training and North-West University for providing funding and facilities to carry out my research.

I owe a huge amount of gratitude the team of Glasgow veterinary School for the help, advice and support, I’m really grateful.

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ABSTRACT

Diarrhoea is a complex condition mostly encountered in small animal practices incriminating some common enteric pathogens including canine parvovirus type 2, canine coronavirus, canine distemper virus, canine rotavirus, endoparasites, bacteria as well as life style and diet. The CPV, CCoV and CRV are the main viral enteric pathogens responsible for severe gastroenteritis including diarrhoea; whereas CPV-2 is the cause of high morbidity and high mortality in dog populations.

Since its appearance in the late 1970, several evolutions and mutations of CPV have been observed, resulting in the replacement of the original type by its new variants 2a, CPV-2b and later on CPV-2c, circulating worldwide. Nowadays, in severe cases of CPV-2 infection, it was found that there is mix infection between CPV-2 and CCoV, or CPV-2 and CRV infection. Therefore, the monitoring of these infectious diseases is critical for control and prevention.

Our study aimed to determine the epidemiology of bloody diarrhoea, to identify and discriminate viral enteric pathogens circulating among puppies presented to the North West University, Animal Health hospital, Mafikeng campus by means of Immunochromatography and molecular techniques.

Faecal samples of 84 diarrhoeic dogs presented with signs suspected to be due to CPV-2 infection were collected at consultation. In addition, other demographic informations were collected for each dog. Two diagnostic methods were used: Immunochromatography assay (IC) for antigen detection and conventional PCR using the universal CPV primers for characterisation. Of the 84 samples, IC tested 66/84, 2/84 and 2/84 positive CPV-2, CCoV and CRV antigens respectively. Conventional PCR revealed 80/84, 0/84 and 12/84 positive CPV-2 DNA, CCoV-RNA and CRV-RNA respectively. The analysis of the sequences confirmed the presence of 80 CPV-2 strains and 12 CRV strains. The predominant circulating variant was the CPV-2c followed by CPV-2b (minor); and G3 rotavirus serotype. The statistical results showed a significance (P<0.05) correlation between the IC and PCR results on CPV-2 and CRV, whereas no significance were observe concerning CCoV. Additionally CRV showed a statistical significance between vaccinated and unvaccinated dogs, whereas CPV and CCoV did not. The phylogenetic analysis performed on CPV-2 and CRV sequences revealed

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similarities with strainsfrom Korean, Peru, China, India, and Nigeria for CPV and Japan for CRV respectively.

The study revealed that predominant positive samples were obtained from puppies aged between 0 – 12 weeks. However, no correlation was found between ages and the occurrence of the disease among patients. There was no significant correlation between sex and the occurrence of CPV virus, neither a correlation between the occurrence of the disease and the location of the dogs. Most of the affected animals had either been vaccinated once or never. In addition, the study noted that some animals had a co-infection of CPV and CRV viruses. In this survey, the characterisation of the CPV-2c and CPV-2b variants, and the one of CRV serotype will contribute to the understanding of the pathogenesis of the diseases and the awareness of monitoring strategies for an efficient control and prevention of infections for a better public health.

In conclusion, this study revealed that the majority of animals presented with gastroenteritis to the Animal Health Hospital at the North West University, Mafikeng Campus were predominantly affected by the CPV virus and mainly the CPV-2 virus.

There is a need of more awareness for pet owners on the issue of vaccination in order to reduce the prevalence of the diseases in the area.

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LIST OF ABBREVIATIONS

Asp: Aspartic Acid

bp: Base Pairs

CAD: Canine Adenovirus

CCoV: Canine Coronavirus

CDV: Canine Distemper virus

CECoV: Canine Enteric Coronavirus

CPE: Cytopathic Effect

CPV: Canine Parvovirus

CRCoV: Canine Respiratory Coronavirus

CRV: Canine Rotavirus

C-PCR: Conventional Polymerase Chain Reaction

DNA: Deoxyribonucleic Acid

ELISA: Enzyme Linked Immunoassay

EM: Electron Microscopy

EtBr: Ethidium Bromide

HCoV: Human Coronavirus

FIPV: Feline Infectious Peritonitis Virus

FPLV: Feline Panleukopenia Virus

GIT: Gastrointestinal tract

Glu: Glutamic Acid

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HA: Haemagglutination

HGE: Haemorrhagic gastroenteritis

HI: Haemagglutination Inhibition

IC: Immunochromatography

IF: Immunofluorescence

IgM: Immunoglobulin M

iiPCR: Insulated Isothermal Polymerase Chain Reaction

kb: Kilo Base

MCL: Maximum Composite Likelihood

MDA: Maternal Derived Antibodies

MGB: Minor Groove Blinders

MLV: Modified Live Vaccine

MVC: Minute Virus of Canine

NS: Non Structural

ORFs: Open Reading Frames

PEDV: Porcine Epidemic Diarrhoea Virus

pH: Potential of hydrogen

PRCoV: Porcine Respiratory Coronavirus

PCR: Polymerase Chain Reaction

QCM: Quartz Crystal Microbalance

qRT-PCR: Real-Time Reverse Transcriptase Polymerase Chain Reaction

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RBC: Red Blood Cells

RFLP: Restriction Fragment Length

Polymorphism rmp: Rounds per minutes

RNA: Ribonucleic Acid

RT-PCR: Real-Time Polymerase Chain Reaction

SNPs: Single Nucleotide Polymorphism

Spp: Species

TGEV: Transmissible gastroenteritis Virus

TAE: Tris acetic acid EDTA

UV: Ultraviolet

VI: Viral Isolation

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LIST OF SYMBOLS

> Greater than

≥ Greater and equal ≤ Less and equal

< Less than ± Plus minus % Percentage ꞵ Beta ℃ Degree Celsuis g Gram mg Milligram nm Nanometre mA Milliampere ml Milli litre µl Micro litre

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LIST OF FIGURES

Figure 2. 1: Classification of CPV ... 5

Figure 2. 2: Representation of the lumen of small intestine.. ... 10

Figure 4. 1: Immunochromatography assay kit to use………40

Figure 4. 2: Illustration of the overall age distribution as a percentage. ... 41

Figure 4. 3: Dogs presenting with gastrointestinal enteritis according to their sex ... 41

Figure 4. 4: Distribution of Dogs breeds presenting with gastroenteritis ... 42

Figure 4. 5: Parasite species found on the dogs that were positive for eggs. ... 43

Figure 4. 6: Vaccination status of dogs with gastroenteritis. ... 44

Figure 4. 7: Area of origin of dogs presenting with gastroenteritis. ... 45

Figure 4. 8: Comparison between the IC Test and PCR results for CPV in all tested samples ... 46

Figure 4. 9: Correlation between dog’s sex and the occurrence of CPV. ... 47

Figure 4. 10: Distribution of the CRV by screening test (IC) and Confirmatory test (PCR) . 48 Figure 4. 11: Distribution of the CRV by screening test (IC) and Confirmatory test (PCR). 48 Figure 4. 12: Distribution of the CCoV by screening test (IC) and Confirmatory test (PCR)49 Figure 4. 13: Occurrence of the CPV among vaccinated and unvaccinated dogs ... 50

Figure 4. 14: Occurrence of CRV among vaccinated and unvaccinated dogs ... 51

Figure 4. 15: Occurrence of CCoV among vaccinated and unvaccinated dog ... 52

Figure 4. 16: Overall percentage of sensitivity and specificity for CPV between IC and PCR ... 54

Figure 4. 17: Overall percentage of sensitivity and specificity for CRV between IC and PCR. ... 55

Figure 4. 18: Overall percentage of sensitivity and specificity for CCoV between IC and PCR. ... 56

Figure 4. 19: The CPV-2 fragments (1-24)... 57

Figure 4. 20: A rooted plylogenetic tree of Mafikeng CPV-2 strains. ... 61

Figure 4. 21: The CRV fragments (1-12) ... 61

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LIST OF TABLES

Table 3. 1: Primer sets for canine parvovirus ... 32

Table 3. 2: The PCR components volume to use for DNA... 33

Table 3. 3: Thermo cycling parameters for PCR amplification of viral DNA from faecal samples ... 33

Table 3. 4: Thermo cycling parameters for PCR amplification of CRV from faecal samples36 Table 3. 5: Thermo cycling parameters for PCR amplification of CCoV from faecal samples ... 36

Table 3. 6: The PCR components volume to use for RNA ... 36

Table 4. 1: Summary of egg distribution among dogs presenting with gastroenteritis ... 43

Table 4. 2: The p-values results of CPV, CRV, and CCoV between IC and PCR test ... 46

Table 4. 3: Statistical comparison of the occurrence of the three diseases among vaccinated and unvaccinated dogs ... 49

Table 4. 4: Relationship between Demographic Variables and the prevalence of CPV and CRV ... 53

Table 4. 5: Viral strains from the PCR products after sequence analysis and their accession numbers in GenBank for CPV-2 ... 58

Table 4. 6: Viral strain from the PCR product after sequence analysis and their accession numbers in GenBank for CRV ... 62

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CHAPTER ONE

INTRODUCTION

Gastroenteritis is a condition in which the stomach and the small intestine are affected in the inflammatory processes (Howell, 1996). Diarrhoea itself is not a disease, it is a complex (syndrome) from pathogens of different origins like bacteria, viruses, parasites, environment; and lifestyle condition like food and weaning (Casseleux, 2009). The acute onset of bloody diarrhoea is the main characteristic of gastroenteritis (Kahn et al., 2005).

Gastroenteritis and/or haemorrhagic gastroenteritis is an important threat to consider in the kennel population as it is caused mainly by canine parvovirus, known for its rapid propagation (Decaro & Buonavoglia, 2012; Kaur et al., 2016); Bloody diarrhea also known as “Strawberry jam” is the typical form of gastroenteritis; mostly with mucus involvement and increased frequency of defecation (Casseleux, 2009).

The most affected in the kennel population are puppies under 6 months of age, mostly between 6-20 weeks of age (Tupler et al., 2012) compared to adult dogs which are less at risk. The younger ages are at high risk to develop gastroenteritis due to the fact that the maternal antibody protection decreases with time and the protection status in puppies, through vaccination, has not yet been adequately established to protect them against the infection (Kahn et al., 2005). Marks et al. (2011); Grellet et al. (2014) described the enteric pathogens involved in diarrhoea in canine populations:

 Canine parvovirus (CPV)  Canine coronavirus (CCoV)  Salmonella Spp

 Campylobacter Spp  Clostridium perfringens  𝛃-haemolytic Escherichia coli  Giardia Spp

 Toxocara Spp  Ancylostoma Spp  Cystoisospora Spp

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The three latter are parasitic enteropathogens; while the most frequently encountered viral enteric pathogens are canine parvovirus (CPV) and canine coronavirus (CCoV) (Marks et al., 2011). These two viruses are very complementary in the way that CPV attacks cells with high replication levels (digestive cells from crypts); while CCoV attacks mature cells (digestive cells located on the apex of villosities) (Casseleux, 2009). The combination of these two viral pathogens explain the fatal diarrhoea observed during weaning in puppies.

Ortega et al. (2017) demonstrated in their study that the co-infection of the CRV and CPV displays a severe condition of viral gastroenteritis because rotavirus has a low mortality rate. However, it remains a threat to global health due of its zoonotic features as compared to CPV which is the main important pathogen of canine gastroenteritis responsible for high mortality in the dog populations.

The risk of death increases with poor food consumption leading to the degradation of faeces quality which is associated with reduced daily weight gain. The dehydration indicates a severe condition; with appropriate supportive care, most dogs recover within a short period (Kahn et

al., 2005). Many cases of diarrhoea resolve with or without symptomatic treatment due to the

fact that its aetiology and factors involved are misunderstood (Stavisky et al., 2011). Laboratory confirmation of the pathogen involved is ideal (Desario et al., 2005). Therefore it is important to extend the understanding of this most frequently reported concern in the kennel field.

In Africa, the prevalence of CPV-2 strains were described by Steinel et al. (1998) demonstrating that CPV-2b was the predominant strain present at 66 % in the southern part of Africa. In South Africa, studies done by Dogonyaro et al. (2013) on CPV-2 variants revealed that the predominant CPV-2 circulating was 2b followed by 2a. Up until now there were no studies reported on CPV-2c on the canine populations except for a case from serval (Leptailurus serval) detected with CPV-2c (Oosthuizen et al., 2019).

1 1. PROBLEM STATEMENT

Several factors can induce bloody gastroenteritis in puppies as a result of mixed effects between 22 pathogens and lifestyle risk factors (Stavisky et al., 2011). Although its aetiology is often incompletely understood. Most of the time, at first sight before any diagnostic procedure done, bloody diarrhoea tend to be confused with parvovirus infection in regard to the clinical signs. However, some tests helps to discriminate parvovirus from other conditions:faecal floatation

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rule out worms and coccidian, wet preps rule out giardia, blood smear with signs of neutrophilia indicate parvovirus and you have the rapid parvo snap test for confirmation. The challenge is mostly to differentiate diarrhoea caused by parvovirus from the one of coronavirus and rotavirus; because all three being involved in the viral infection induction.

Therefore it is important to identify and determine the causative pathogen involved in bloody diarrhoea in puppies that are presented to the Animal Health Hospital, Mafikeng Campus, North-West University; although undocumented in Mafikeng.

1 2. THE STUDY AIM

The aim of the present study was to determine the aetiology of bloody diarrhoea and identify which potential aetiological viral pathogens are circulating among puppies brought to the Animal Health Hospital, Mafikeng Campus; by means of Immunochromatography assay as a screening test and molecular method in the laboratory for confirmation.

1 3. OBJECTIVES OF THE STUDY

The objectives of the study were:

 To establish the viral causes of bloody diarrhoea in puppies presented at the Animal Health Hospital, Mafikeng campus using faecal floatation rule out worms and coccidian and the rapid parvo snap test for confirmation.

 To determine the viral aetiology of bloody diarrhoea using conventional PCR.

 To determine the reliability of the rapid test (snap test) compared to the molecular technique of conventional-PCR, in order to compare if the results obtained from rapid test are uncontestable with those of molecular technique.

1 4. JUSTIFICATION

The clinical manifestations of bloody diarrhoea tend to be confusing sometimes with those of CPV infections in puppies. In clinic, puppies presented with bloody diarrhoea present several signs of parvovirus infection that can be in contradiction with the results of the snap test; which became a dilemma practically. So the molecular approach remains the ideal way to determine the final diagnosis of the bloody diarrhoea (suspected as parvovirus) as this can be caused by the three viruses of the gastrointestinal tract (GIT) (Parvovirus, Coronavirus and Rotavirus).

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CHAPTER TWO

LITERATURE REVIEW

2.1. INTRODUCTION

Gastroenteritis is a medical term referring to the inflammation of the (GIT), usually the stomach and intestine (Howell, 1996). Haemorrhagic gastroenteritis (HGE) is a very serious condition affecting dogs and is characterised by an acute onset of bloody diarrhoea in a formerly healthy dog (Kahn et al., 2005). The typical sign is a sudden onset of profuse bloody diarrhoea with a foul odour affecting young dogs age between 2-4 years old (Willard, 1983). HGE is an important threat to consider in the kennel population as its propagation and spread is usually rapid, mainly due to the role played by CPV in the process (Decaro & Buonavoglia, 2012; Kaur

et al., 2016).

Diarrhoea is not a disease, it is a clinical sign that can have several infectious causes; and it is known as one of the concerns reported most frequently in dogs (Casseleux, 2009). It is also understood as a change observed in the bowel’s movement that affects certain parameters such as frequency, fluidity, content and volume. Its aetiology is unknown as it is a multifactorial condition in dogs (Stavisky et al., 2011).

Some enteric pathogens are considered involved in diarrhoea and gastroenteritis in canine population, namely:

 Canine parvovirus (CPV)  Canine coronavirus (CCoV)  Canine rotavirus (CRV)  Canine distemper (CDV)  Salmonella Spp.

 Campylobacter Spp.  Clostridium perfringens

 𝛃- haemolytic Escherichia coli (Marks et al., 2011)

The most common viral pathogens of the GIT are CPV, CRV and CCoV; Giardia Spp.,

Toxocara Spp., Cystoisospora Spp., Ancylostoma Spp. are parasitic enteropathogens which can

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history, the diet and the contact between species also contribute to acute diarrhoea cases (Stavisky et al., 2011).

Canine parvovirus, Canine rotavirus, Canine distemper virus and Canine coronavirus represent the four families of viruses aetiologically responsible for severe enteritis in dogs (Pollock & Carmichael, 1982). Therefore, this study will mostly focus on the gastrointestinal viruses known to be the most expected in the kennel population, responsible for diarrhoea named: CPV, CCoV and CRV (Casseleux, 2009) as they are most difficult to diagnose.

2.2. DESCRIPTION AND GENERAL ASPECT OF VIRUSES

2.2.1. CANINE PARVOVIRUS

Canine parvovirus type 2, the main enteric pathogen in dogs is a small, non-enveloped, single stranded DNA genome with an icosahedral capsid of about 25 nm of diameter (Appel & Barr, 2009). CPV-2 belongs to the family of Parvoviridae (Strassheim et al., 1994), of the genus Protoparvovirus, together with feline panleukopenia virus. The Figure 2.1 illustrates the classification of CPV-2 (Tattersall & Cotmore, 1990).

Infect Vertebrates Infect Insects

Parvovirus Erythrovirus Dependovirus

Densovirus Iteravirus Contravirus

Figure 2. 1: Classification of CPV

The origin of CPV has not been established, but it was demonstrated that it is a very stable virus with the ability to withstand high temperature and any pH (Kahn et al., 2005). Resistant to heating (60 ℃ for one hour), resistant to pH 3 and ether treatment (Willard, 1983).

Family Parvoviridae

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In nomenclature, there are two types of canine parvovirus viruses: canine parvovirus type I (CPV-1) and canine parvovirus type II (CPV-2). Canine parvovirus type I (CPV-1) also called minute virus of canines (MVC) has been identified as the cause of neonatal death in dogs (Carmichael, 1994); additionally it is incriminated as causing myocarditis in neonatal puppies (Hayes et al., 1979). Recently, it has been shown that CPV-1 is unrelated genetically and antigenically to the viruses of the genus parvovirus belonging to the Parvoviridae family. However, it is considered as part of the genus Bocavirus together with bovine parvovirus and human bocavirus (Tattersall et al., 2005).

Canine parvovirus type 2 (CPV-2) known as the original viral strain for CPV infection, causes severe, fatal haemorrhagic gastroenteritis in 2 to 6 months old puppies (Decaro et al., 2006a; Truyen, 2006). CPV-2 appeared as a canine pathogen in the late 70s. It is believed to be derived from feline panleukopaenia virus (FPLV) described as a cat infection and presents the same resemblance with the latter (Truyen et al., 1994).

Canine parvovirus type 2, discovered in the late 1970, has mutated and evolved leading to the appearance of three antigenic variants: CPV-2a, CPV-2b and CPV-2c (Parrish et al., 1985). In 1980, CPV-2a and CPV-2b were described as new mutant antigenic variants and replaced the original CPV-2. The third variant, CPV-2c appeared later in the 2000s and was discovered in Italy and predominantly circulating in European countries (Buonavoglia et al., 2001) and distributed in Asia and America (Truyen, 2006). All three variants happened to be actually more dominant and prevalent worldwide in canine populations (Decaro et al., 2006b; Kaur et

al., 2016) as they have totally replaced the original CPV-2 (Buonavoglia et al., 2000) known

to be the source of high morbidity and mortality in young dogs (Goddard & Leisewitz, 2010). Two different forms associated with CPV-2 infection are observed:

 Enteritis  Myocarditis.

The difference between the original CPV-2 and its antigenic subtypes resides at the position 426 of the VP2 capsid protein whereas 5-6 amino acid of the CPV-2 has mutated and consequently gave rise to the emergence of CPV-2a and CPV-2b (Parrish et al., 1991). However, CPV-2a differs from CPV-2b by the presence of 2 single nucleotide polymorphisms (SNPs) in the gene sequence of the VP (Decaro et al., 2005a). Therefore, CPV-2a is now termed Gln 426; CPV-2b is termed Asp-426; CPV-2c named Glu-426 mutant and was found in adult vaccinated dog with severe gastroenteritis (Decaro et al., 2008).

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The DNA genome is composed of 2 open reading frames (ORFs) in which one plays a role in the viral DNA replication and transcription to encode non-structural proteins (NS1 and NS2); the other one plays a role in encoding the capsid proteins of the virus (Pérez et al., 2007). The viral protein (VP) comprise VP1, VP2, and VP3 where VP2 structure the non-enveloped icosahedral capsid of CPV-2 (Mochizuki et al., 1993; Strassheim et al., 1994).

Regardless of the availability of safe and efficacious vaccines worldwide, CPV-2 constitute the major cause of serious and often fatal disease in dogs populations (Ling et al., 2012). Together with CCoV, they are mainly responsible for acute gastroenteritis (Decaro et al., 2006a).

2.2.2. CANINE CORONAVIRUS

Corona viruses are large enveloped, single stranded, positive-sense RNA viruses with a genome of 27-31 kb; they belong to the family of Coronaviridae, order Nidovirales, genus

Alphacoronavirus (De Vries et al., 1997; Decaro et al., 2011; Adams & Carstens, 2012).

Morphologically, coronaviruses are seen as spherical, enveloped viruses with large club-shaped surfaces. Coronaviruses are divided into 3 antigenic groups (I, II, III) whereas CCoV is classified within the group I coronaviruses as well as FIPV (Feline infectious peritonitis virus), Feline coronaviruses, TGEV (Transmissible gastro-enteritis virus of swine), PEDV (porcine epidemic diarrhoea virus), PRCoV (porcine respiratory coronavirus), and human coronavirus (HCoV-229E and HCoV- NL63) (Decaro & Buonavoglia, 2008; Patel & Heldens, 2009). With reference to the order Nidovirales, the word nido from the Latin Nidus meaning nest, which imply to the 3’ co-terminal nested subgenomic viral mRNA that is produced during replication of the virus (Balasuriya & Stott, 2004). It is noted the existence of 2 categories of CCoV (Decaro & Buonavoglia, 2008): canine enteric coronavirus (CECoV), known as CCoV belongs to the group I coronaviruses, and canine respiratory coronavirus (CRCoV) belonging to the group II coronaviruses. CECoV is the cause of moderate to severe gastroenteritis in puppies (Decaro et al., 2004); although divided in 2 distinct genotypes identified in the faeces of infected puppies with enteritis (Pratelli et al., 2004):

 CCoV type I (CCoV-I)  CCoV type II (CCoV-II).

Canine coronavirus has an envelope that contain 2 viral structural glycoprotein spikes S and M, the small membrane and the nucleocapsid N. The glycoprotein S forms the external membrane plays a role in neutralising antibodies and is responsible for binding virions to the

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host cell membrane; whilst the glycoprotein M forms the transmembrane is lodged in the envelope (Enjuanes et al., 2000). Additionally, the small membrane constitutes the assembling envelope; and the nucleocapsid N constitutes the helical protein that plays a role in modulating the synthesis of viral RNA (Enjuanes et al., 2000).

Canine coronavirus are inactivated by lipid-solvents with a stable pH of 3.0; heat-labile; this virus is still virulent in cold conditions. Though it plays a role in tracheobronchitis (Kennel-cough) in dogs as demonstrated recently by some studies (Balasuriya & Stott, 2004).

2.2.3 CANINE ROTAVIRUS

Rotaviruses belong to the family Reoviridae, genus rotavirus. Canine rotavirus is a non-enveloped, icosahedral capsid, triple layered virus with a double-stranded RNA; which is approximately 60-75 nm in diameter (Martella et al., 2010). The virus is grouped into 8 species (A-H) whereas the genome is composed with 11 fragments of RNA that encodes 12 viral proteins, 6 structural proteins and 5 or 6 non-structural proteins (Matthijnssens et al., 2012). Morphologically, the virus has the shape of a wheel, best identified using electron microscopy. The outer viral capsid proteins VP4 and VP7 form the platform on which the dual system of classification for rotaviruses is based (Martella et al., 2001). The outer capsid proteins (VP4 and VP7) are implicated in viral neutralisation; whereas VP4 is the minor neutralising antigen referred as the P type, and VP7 is the major neutralising antigen referred to as the G type (Estes, 1996; Desselberger, 2014). Therefore, VP4 is termed protease-sensitive P-type and VP7 termed glycosylated G-type (Iturriza-Gómara et al., 2004). Serological and genomic techniques are then implemented for the differentiation within types, subtypes and serotypes (Martella et al., 2001). In addition, for VP4, another particular identification has been used for ease of reference for P serotypes (open numbers) and P genotypes (numbers in brackets) (Estes & Cohen, 1989). Rotaviruses are known as the most important gastroenteric pathogens causing acute watery dehydrating diarrhoea in both humans and various animals species (Greenberg & Estes, 2009); and they are considered as zoonotic agents (Martella et al., 2010).

2.3. PATHOGENESIS AND TRANSMISSION OF VIRUSES

The faecal oral route is the principal way of transmission of CPV. The infection is spread through oronasal exposure or faecal ingestion by direct contact with contaminated faeces or

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indirect contact with contaminated objects, soil and environment (Decaro et al., 2005b). The intestinal crypts and lymphoid organs are the target tissues for CPV replication (Pollock, 1982). Once the virus penetrates through oronasal route, the retropharyngeal and mesenteric lymph nodes (gastroenteric-associated lymphoid tissues) are the first site for replication (Potgieter et

al., 1981). The virus remains in the serum for five days after infection, then it is disseminated

in the body through bloodstream and thereafter transported by infected leucocytes to the germinal epithelium of the crypts of the small intestine with resultant diarrhoea (Pollock, 1982). Infected puppies shed the virus in the faeces after an incubation period of four to five days prior to exposure and 10 days maximum post recovery (Decaro et al., 2005a). High titres are observed in the faeces of infected dogs (Pollock, 1982) especially type 2a and type 2b (Carmichael, 1994), whereas in the tonsils and intestinal tissues are found the prominent viral titre (Meunier et al., 1985). In puppies that died as a result of CPV infection, gross lesions such as haemorrhagic enteritis of the small intestine and enlargement of mesenteric lymph nodes and Peyer’s patches are identified (Greene and Decaro, 2012); villous atrophy; and dilation of crypts which have no epithelial cells (Azetaka et al., 1981).

The incubation period for CCoV is 1-4 days, and the viral replication occurs in the epithelial cells of the large intestine where the virus destroys the absorptive mucosa, thus desquamation and atrophy of the colonic ridges and therefore shortening of the intestinal villi with consequence diarrhoea as a result of malabsorption and indigestion (Balasuriya & Stott, 2004). Canine coronavirus has a high morbidity and low mortality rate, typically self-limited to the GIT (Decaro & Buonavoglia, 2008; Tennant et al., 1991) and more prone to infect sheltered and kennelled dogs (Pinto et al., 2014). The genetic evolution of a more virulent strain known as CB/05 variant tends to aggravate the disease and accentuate the high mortality (Patel & Heldens, 2009).

Although, the high mortality rate encountered is due to a combination or synergistic effect or mixed infection with other pathogens like CPV, CDV and/or canine adenovirus type I (Pratelli

et al., 2001b), the consequence of which whether the infection will be mild or severe (Appel,

1988). Dogs shed the virus for 2 weeks or longer; therefore the contamination is via faecal-oral route, and the virus is transmitted via inhalation or aerosols and ingestion through saliva (Patel & Heldens, 2009).

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Martella et al. (2001) demonstrated that group A rotaviruses, considered as the most important pathogens are mainly responsible of neonatal diarrhoea in both humans and several animals species; and the condition is often worsened by a lack of hygiene and poor management (Clasen

et al., 2014). Rotavirus infection is not really seen as a pathogenic condition of dogs and cats,

as the infection is usually subclinical with mild enteritis (Cook et al., 2004). However, the infection induced does not have great impact as the disease is minor in puppies. In addition, it has been noticed that puppies less than 12 weeks of age happen to be more sensitive to the infection and exhibit a diarrhoea within 20-24 hours (Johnson et al., 1983; Guirao, 2009).

Figure 2. 2: Representation of the lumen of small intestine. Illustration of the predilection sites

for the pathophysiology of canine parvovirus, canine rotavirus and canine coronavirus (Pratelli, 2006).

2.4. CLINICAL SIGNS OF VIRUSES

Carpenter et al. (1980) reported that the most evident clinical signs for CPV-2 infection is enteritis; usually accompanied by a rapid and sudden onset of diarrhoea, vomiting, dehydration, anorexia and a well-marked swollen abdomen, depression, and death as early as two days after the onset of the disease (Carpenter et al., 1980). Additionally, apparent signs like bloody

 Coronavirus

 Rotavirus

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diarrhoea with a foul smell, fever ≥ 39.4 ℃, leukopaenia, and neonatal death due to myocarditis in puppies (Yilmaz et al., 2005) are all part of the symptoms.

Parrish (1995) demonstrated that the clinical manifestation of CPV infection depend on the age of the host; while Decaro et al. (2005a) reported that the clinical form of the disease is characterised by an haemorrhagic diarrhoea. Decaro et al.(2009a) observed that haemorrhagic gastroenteritis and leukopenia are the common clinical signs associated with CPV infection. Prittie (2004) reported that CPV enteritis presented non-specific clinical signs characterised by anorexia, depression, and fever; thus typically limited to a severe GIT disturbance and immunity suppression. Smith-Carr et al. (1997) reported that vomiting is the initial manifestation of the condition in puppies followed by small bowel diarrhoea 24-48 hours after the onset of clinical signs. Additionally, Pollock and Coyne (1993) reported leukopenia in severe cases, lymphopenia and an acute onset of enteritis.

Greene and Decaro (2012) reported that haemorrhagic diarrhoea, vomiting, fever, lymphopenia and death in certain circumstances, constitute the signs of CPV infection. Ortega et al. (2017) described the clinical signs of CPV infection to include fever, anorexia, lethargy, depression, vomiting, mucoid to haemorrhagic diarrhoea and sometimes leukopenia.

Clinical signs are mild, often asymptomatic and include loose to watery faeces, often with mucus or blood, anorexia, dullness, vomiting and diarrhoea for ± 2 weeks (Tennant et al., 1991); and the disease is highly contagious (Carmichael & Binn, 1981). Anorexia, vomiting, dehydration, soft to fluidly diarrhoea are the clinical signs of CCoV described by Pratelli (2006).

2.4.3. CANINE ROTAVIRUS

There is no pathognomonic clinical signs for CRV infection as the disease is subclinical and the infection asymptomatic; mostly confusing with signs of CPV infection or CCoV infection suggesting a possibility of co-infection and a mixed-effect between pathogens (virus, bacteria, parasites) (Martella et al., 2001; Yilmaz et al., 2007; Solberg et al., 2009).

It has been observed that Rotavirus infection causes severe dehydrating diarrhoea mostly in infants younger than 5 years of age and in puppies younger than 2 weeks of age; associated with anorexia and vomiting, often fatal (Greenberg & Estes, 2009).

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Ortega et al. (2017) described the clinical signs of rotavirus infection as mild enteritis especially in pups below two weeks old; additionally there may be lethargy, anorexia, fever, diarrhoea and vomiting. Pollock and Carmichael (1990) reported that CRV infection is less important in pups as usually the most affected pups are younger than 2 weeks old. The signs are subclinical and a mild form of enteritis associated with anorexia and vomiting.

2.5. IMMUNITY AND CONTROL OF VIRUSES

Ling et al. (2012) reported that CPV-2 infection was identified as a disease affecting mostly young dogs under 6 months of age; with a high mortality occurring in puppies less than a year old. This can be explained by the interference between vaccine antigen and antibodies from the first immunity acquired at birth, through colostrum to provide protective immunity in new-borns (Ling et al., 2012).

The interference between maternal derived antibodies (MDA) from vaccinated dams to puppies (via colostrum) and the CPV vaccine itself constitute the major problem of CPV vaccination failure (Decaro & Buonavoglia, 2012). For puppies less than 2 weeks of age, who did not acquired enough MDA, it’s not recommended to vaccinate with MLV core vaccine as it can cause the disease and predispose to death (Day et al., 2016). Moreover, the immune system is not well balanced during the early time after birth or during the first week, because of the thermoregulation default.

The ideal goal and best way to control and prevent CPV-2 in domestic animals is through immunization (Pollock & Carmichael, 1983; Nandi et al., 2013). The adequate approach is to vaccinate only after waning of MDA, so that active immunisation will be freely acquired (Greene & Decaro, 2012), mainly using a modified live virus vaccine (MLV) (Decaro & Buonavoglia, 2012). Other vaccines like inactivated vaccines are less effective and take much longer to induce an immune response and induce only short-term immunity (Truyen & Parish, 2013).

Previous studies demonstrated that most of the vaccines used currently, especially the modified live virus vaccine are made with CPV-2 or CPV-2b, thus effective to induce protection against all variants including CPV-2c (Decaro & Buonavoglia, 2012; Wilson et al., 2013). Nevertheless, the prevalence of CPV-2 infection is still a challenge as some young and adult dogs displayed clinical signs of CPV infection despite the completion of a full vaccination

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calendar as demonstrated by Decaro et al., 2008; Decaro et al., 2009b; and Mylonakis et al., 2016.

Truyen (1999) observed that the standard vaccines used nowadays incorporate an inactivated and a MLV vaccine to prevent against CPV infection. Additionally, the evolution and the apparition of CPV-2 new variants make these vaccines ineffective for the protection of puppies against the infection.

Day et al. (2016) reported that CPV-2 variant is still present in some of the current MLV vaccine used, therefore stimulates an active immune response that provide protective immunity of ≥ 4 years against all variants including CPV-2c. Additionally, Day et al. (2016) reported that inactivated (killed) vaccines are not beneficial in routine use as they are only administered where MLV are not recommended like in pregnant bitches, wild and exotic species. Thus, inactivated (killed) vaccine are less effective and take long time to induce an immune response (Day et al., 2016). Although, puppies less than 4 weeks of age can’t be vaccinated because the immune system is protected by the MDA and vaccination with MLV is inappropriate, as it can trigger the disease and some pathologies (Day et al., 2016).

Altman et al. (2017) reported that puppies vaccinated later than 12 weeks have their risk of vaccination failure decreased; and the use of final vaccination at less than 12 weeks of age predispose to vaccination failure. Furthermore, a review for the final vaccination age for puppies to be at/or after sixteen weeks, as the age of administration for last vaccination plays an important role in vaccination failure (Altman et al., 2017).

Smith-Carr et al. (1997) reported that the most evident and incriminating factors in the occurrence of CPV infection are a lack of protective immunity (due to the non-acquisition or non-receptivity of passive transfer of antibodies from dams to puppies through colostrum), incomplete or ineffective primary CPV vaccination course and/or failure of vaccine to provide and induce immunity.

Prittie (2004) demonstrated that natural infection of CPV always presents a high positive value on IC or ELISA as compared to an infection induced by vaccination of maternal antibody which will have a probability to be a false positive. Several authors also demonstrated that due to the fact that CPV-2 shedding period is short (2 weeks), vaccination with live attenuated CPV-2 vaccine may induce a false positive result of IC or ELISA few days post vaccination.

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Khan et al. (2006) reported that CPV-2 infection is mostly observed in unvaccinated dogs for reasons like poor management and hygiene, cost of vaccines, and often unawareness of pet’s owners. Calderón et al. (2011) explained that to manage CPV-2 infection in little puppies, it is better to vaccinate pregnant bitches in order to increase the level of specific colostral and milk antibody in dogs.

The composition of the vaccine currently used in the control and vaccination of CPV infection worldwide is basically made with the original type 2 as the antigen or its variant; so that puppies are fully and efficiently protected from the disease and the infection (Decaro & Buonavoglia, 2012).

Decaro and Buonavoglia, (2012) stated that primary reasons for the fast expanding of CPV-2 regardless vaccination are either a poor immune system or the interference of MDA in vaccinated puppies. However, in South Africa, the disease is mostly related to non-vaccination causes. As stated earlier, because the level of MDA interfering with CPV vaccination causing inefficacity of the vaccine, high titre CPV vaccine works adequately to protect puppies from this dilemma (Kahn et al., 2005).

Several studies (Pérez et al., 2007; Decaro et al., 2007) clarified that the original subtype 2 is no longer circulating and has been replaced by its variants, especially the latest one CPV-2c. The CPV-2 as the antigen of the vaccine currently used is still suitable to cover a long term immunity protection against all subtypes (Spibey et al., 2008). Although there are studies currently to evaluate whether the new antigenic variant (CPV-2c) can prevail adequately in the full protection of the puppies without interference. As a results a potential cross- protection might be observed after vaccination (Wilson et al., 2014).

The role played by CPV variants during their evolution and antigenicity has shown that CPV is really posing a sanitary security problem worldwide; so many studies and researches gathered some preventives measures and strategies to help assist in the control of CPV spread (Nandi et al., 2013):

 Immediate isolation of sick puppies and/or adults

 Good hygiene and instructions to the working team about the risk of contamination, so that regular disinfection of instruments, area and personnel has to be followed.

 Quarantine of 14 days for all sick or exposed animals and 30 days for all newly introduced animals.

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 Grouping of animals according to age; separate young from adults

 The use of hand sanitizers with 70% alcohol (alcohol does not kill the virus, but the bacteria and other pathogens).

 Vaccination and deworming protocols and schedules to be followed.

Spibey et al. (2008) investigated whether the CPV-2 live attenuated vaccine (Norbivac intervet) can protect dogs from the virulent type 2c virus. The results of their study showed that vaccinated dogs were not only protected from the clinical aspect of the disease as CPV-2 vaccine-based administration prevented the shedding of CPV-2c virus. Therefore the study reported that vaccination with type 2 vaccine would fully protect all current CPV variants and would build a strong immune response to CPV infections.

There are also other aggravating factors and risk predisposing factors along with CCoV co-infection in the manifestation of parvoviral disease; these include weaning, overcrowding, endoparasite load (Goddard & Leisewitz, 2010; Kalli et al., 2010). Additionally, the sex, breed, age, vaccination status, and the season of the year appear to be influencing factors; young ages, unvaccinated animals were more likely to be CPV positive compare to vaccinated animal (Miranda et al., 2015).

Moskvina and Ermolenko (2016) reported that a number of canine helminths including

Toxocara canis, Ancylostoma caninum, Dipylidium caninum, and Echinococcus species are

considered as zoonotic parasites worldwide and pose a threat to public health. The study also added that the control of these helminths in the environment is complicated by the increased number of stray dogs, and along with the increased number of strays will be the elevated chances of encountering contaminated faeces.

Carmichael & Binn (1981) reported that all breeds and ages are prone to CCoV infection. Ntafis

et al. (2013) reported that older animals are less susceptible to CCoV than younger animals, as

older animals have built immunity from exposure to the infection at an earlier age.

Pollock (1982) reported that there were no effective vaccines available against CRV and CCoV; nevertheless Pratelli (2006) reported that the spread of CCoV infection was difficult to control as the virus is shed for a long period (± 6 months after the clinical signs have disappeared) becoming highly contagious especially when it remained in the environment.

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Therefore the study suggested that the effective control of CCoV requires the prevention of the infection.

Pollock (1982) reported that there were no effective vaccines available against CRV and CCoV.

2.6. DIAGNOSIS OF CANINE VIRAL DIARRHOEA

Several authors (Mochizuki et al., 1993; Uwatoko et al., 1995; Desario et al., 2005) have described methods that could assist in the diagnosis of viral pathogens from faecal samples of diarrhoeic dogs.

There are traditional methods (immunochromatographic (IC), haemagglutination (HA), viral isolation (VI), electron microscopy (EM), and others), that constitute the screening tests or preliminary tests. These tests or techniques need confirmation using laboratory techniques or molecular assay (i.e. conventional polymerase chain reaction (PCR) , real-time PCR, nested PCR, and others) as demonstrated by Desario et al. (2005). Traditional methods focused on detecting viral antigens whilst molecular methods focused on the nucleic acid ̶ based method; as stated by Decaro and Buonavoglia (2012).

Desario et al. (2005) reported that an early and rapid diagnosis is crucial and delicate in the way that it will help prevent the spread of contamination by isolating those presenting clinical signs; and the risk of secondary infection among susceptible animals (Desario et al., 2005). As presented by Stavisky et al. (2011), diarrhoea in dogs results from complex pathogen life-style risk factors and involves multifactorial aspects; therefore, diagnosis made on clinical signs alone is not substantial. Laboratory diagnosis testing is ideal for confirmation of the infection. Decaro and Buonavoglia (2012) observed in their study that traditional methods (IC, HA, VI, EM, and others) have proven to be inferior to molecular techniques in relation to the sensitivity of the results. Whereas the IC assay, compared to molecular technique (nucleic acid – based method) presents a sensitivity not exceeding 50% but the specificity was 100%, usually responsible for a report of high number of false negative antigen test results. Schmitz et al. (2009) observed in their study that the antigen-detection kits present high specificity and low sensitivity results of 18.4%.

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There are a number of laboratory tests and methods to detect CPV-2 in faecal samples of enteric dogs, such as: electron microscopy (EM), ELISA, virus isolation, immunochromatographic test (IC), haemagglutination test (HA), haemagglutination inhibition test (HI), conventional polymerase chain reaction (C-PCR) and real-time polymerase chain reaction (RT-PCR) (Desario et al. (2005).

Electron microscopy (EM)

Electron microscopy is a traditional method of diagnosis by mean of visualizing smaller object and identifying them according to their morphology. Therefore, the detection of CPV-2 in the faeces of dogs (puppies) with gastroenteritis will rely on the diameter size of the viral particles (±20 nm) and the icosahedral shape using a negative stain (Amo et al., 1999; Kaur et al., 2016). The big advantage of using EM in the detection of CPV-2 is the capacity to visualize the virus, to identify it and to confirm its presence; but the disadvantage is that it is required around 106 virus particles per ml to detect CPV-2; that makes its sensitivity very low (Esfandiari & Klingeborn, 2000).

ELISA (Enzyme Linked Immunoassay)

ELISA is a serological test performed to detect specific viral antibodies in the serum samples. Indirect ELISA (also called Sandwich method) is the test performed to detect CPV infection, by mean of IgM antiviral antibody; particularly using a double antibody sandwich ELISA test (Rimmelzwaan et al., 1991). Proksch et al. (2015) reported that false negative faecal antigen ELISA results might be caused by the mutation of CPV strains in the field, not detectable by the faecal antigen ELISA.

Viral isolation (VI)

Viral isolation is a traditional method of diagnosis for detection of CPV-2 that requires acquiring cell lines cultivated in laboratory. Therefore, viral isolation is best achieved with a capable, specialized skilled personnel and availability of cell lines (Desario et al., 2005). Incubation period is 5-10 days, thus time consuming and another step testing by immunofluorescence or HA for the antigen detection; therefore it is a long process (Decaro & Buonavoglia, 2012). A positive result to viral isolation is the production of cytopathic effect (CPE) in the cell culture; or noticeable intranuclear inclusion bodies (Decaro et al., 2005a). Although, Desario et al. (2005) have demonstrated that viral isolation can detect CPV-2 in an

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experimental infection some days post-infection, Viral isolation presents a main disadvantage of low sensitivity (Mochizuki et al., 1993).

Haemagglutination test (HA)

Haemagglutination assay, only done in specialized laboratory, is a traditional method of diagnosis based on the quantification of the virus (with the property to agglutinate) by using red blood cells (RBC) of porcine origin mostly (Desario et al., 2005). The main haematological feature (property) of CPV-2 is its ability to agglutinate erythrocytes on their surfaces from other different animals apart from the pig; but this presents a disadvantage as a large quantity of fresh erythrocytes is required and it is a challenge to obtain. Haemagglutination present the advantages of being simple, rapid and easy to perform and results are available within four hours; but the disadvantage is the low sensitivity and only a well-equipped and specialized laboratory is required (Desario et al., 2005).

Immunochromatography test (IC)

Traditional method of diagnosis known as a SNAP® rapid test performed by professionals (technicians and veterinarians) as well as by patient owners because of the simplicity and rapidity of the procedure (Esfandiari & Klingeborn, 2000). It requires the use of SNAP® Kit containing the antigen of the virus, available on the market and present the advantage of being used in clinical practice or anywhere as a rapid diagnostic test. Its disadvantage is that the result might be biased due to manipulations such as the subjectivity of the test operator, equipment defaults and/ or environmental factors(Desario et al., 2005) and has a low sensitivity (Schmitz

et al., 2009).

Immunochromatography assay is a rapid and safe diagnostic test that detects viral antigen by means of antibody based method also referred as a rapid faecal antigen ELISA test (Desario et

al., 2005). False negative results are very frequently observed and might mislead the

practitioners in the diagnosis and treatment (Schmitz et al., 2009).

Molecular characterisation (PCR)

Polymerase chain reaction assay is a molecular method of diagnosis based on the molecular identification and determination of the viral genome in the faeces of diarrhoeic dogs (Mochizuki et al., 1993). Decaro et al. (2005b) described PCR, especially RT- PCR as a more sensitive, more specific and more reproducible method of diagnosis of CPV-2; whilst Kang et

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means of identifying CPV-2 in the faeces targets the detection of the DNA sequence in the faeces (Buonavoglia et al., 2001).

Several authors have demonstrated that there are different PCR- based methods available in the line of detection of viral antigens:

 Conventional PCR (Mochizuki et al., 1993)  Nested PCR (Hirasawa et al., 1994)

 RT-PCR (real-time PCR) based on TaqMan probe or minor groove binders (MGB) (Decaro et al., 2005b).

Conventional PCR focus on the sequence analysis of fragments of the VP2 by using primers, forward and reverse to encode informations of the amino-acid (Desario et al., 2005; Mochizuki

et al., 1993); Nested PCR uses a double-nested primer pair (inner primer pair) to detect DNA

on agarose gel electrophoresis (Hirasawa et al., 1994); RT-PCR based on TaqMan and Minor groove binders detect and quantify CPV-2 nucleic acid in few hours (Decaro et al., 2005b). However, RT-PCR is not commonly used in most veterinary practice due to the fact that there is a necessity of using specialised materials, reagents and employing highly qualified personnel and the cost is rather prohibitive (Desario et al., 2005).

The diagnosis of CCoV in the laboratory is done by means of electron microscopy, serum virus neutralisation and ELISA test, and PCR for detection of the virus in the faeces (Balasuriya & Stott, 2004). Pratelli (2006) reported that laboratory confirmation is crucial for the diagnosis of CCoV virus; thus the detection in the faeces used EM, isolation in cell cultures and RT-PCR. The detection of CCoV antibodies in the sera required virus neutralisation tests and ELISA.

Mijatovic-Rustempasic et al. (2016) have described several methods in the detection of rotavirus A in the faecal samples, including viral isolation in cell culture, electron microscopy (EM), enzyme immunoassay, coupled reverse transcriptase and PCR amplification (RT-PCR), and real-time reverse transcriptase-polymerase chain reaction (qRT-PCR).

Fitzgerald et al. (1995) reported that G3 serotypes rotaviruses have been found in different species including humans, monkeys, dogs, cats, horses, rabbits, mices, sheeps and pigs. Martella et al. (2001) have described and confirmed that G3 is the unique serotype within the

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canine rotaviruses. Martella et al., (2001) reported that the isolation of canine rotavirus from dogs affected with gastroenteritis is less documented.

Most of the methods of diagnosis described earlier are not quantitative except for RT-PCR (Decaro & Buonavoglia, 2012) as it quantified the virus load shed from the onset of the infection in dogs and carriers (Kumar & Nandi, 2010).

2.6.4. REVIEW OF EPIDEMIOLOGY STUDIES

In previous studies done by several authors on the epidemiology, detection, and characterization of the three viruses (CPV, CCoV and CRV), some had used IC as a screening test and later molecular assay for confirmative diagnosis, as mentioned below.

Schunck et al. (1995) used a touch-down PCR for the detection of CPV and FPLV in the faeces of diarrhoeic dogs, in comparison with another conventional method of diagnostic EM which identify the morphology of the virus particles. The study detected that all the samples identified through electron microscopy were also positive by PCR; some samples were positive by PCR but not by EM as the sensitivity of PCR was more compared to EM. EM is quick but the cost of equipment and maintenance are high. They also reported that touch-down PCR display a more efficient amplification.

Uwatoko et al. (1995) used in their study PCR as a rapid and specific assay to diagnose and identify CPV in the faeces of diarrheic dogs by utilising a double set of primers to amplify a specific 226-bp sequence. They also noted that ELISA test or culture method detected the virus from faeces containing more than 106 PFU/g fresh faeces within 1h, whilst PCR assay combined with the gel filtration was able to detect the virus from faeces containing as little as 103 PFU/g fresh faeces within 3h. This test demonstrated that while the use of ELISA kit is less expensive and more rapid than PCR assay; PCR is able to identify fewer particles of CPV in the environment. Therefore Uwatoko et al. (1995) study is in agreement with the fact that PCR assay is a rapid, specific and sensitive method for detection of CPV from faecal samples. Breed predisposition also plays a big role in the incidence of canine parvovirus enteritis infection as reported by Houston et al. (1996) that American pit bull terrier, Rottweiler, German shepherd dogs, Doberman pinschers are highly at risk compared to small breed like Toy poodles and Cocker spaniels. Additionally the study reported that sexually intact males are twice as likely as sexually intact females to develop CPV enteritis.

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Decaro et al. (2005b) developed a real-time PCR assay for rapid detection and quantification of CPV-2 DNA in the faeces of dogs with diarrhoea; the assay was based on the TaqMan technology. In the study, PCR products were measured by an increase in the fluorescence of the probe by the DNA polymerase used for amplification. The study also demonstrated that negative samples from HA were detected to have CPV-2 DNA and positive samples by HA contained high amount of CPV-2 DNA using this real-time PCR. This finding was in agreement with other studies done previously demonstrating that HA has low sensitivity compared to molecular method.

Decaro et al. (2006c) used minor groove binder probe technology to characterise CPV-2 subtypes in 414 diarrhoeic samples of dogs and reported that the two minor groove binders used have shown to have high specificity, high sensitivity and very reproducible properties ensuring the quantification of CPV-2 DNA with precision compare to the viral DNA load in TaqMan-based PCR assay. Therefore, the study has demonstrated that MGB probe assay is an attractive tool, reliable for identification and rapid characterisation of CPV variants.

Pérez et al. (2007) conducted a study in puppies aged 1-11 months old (vaccinated and unvaccinated) with haemorrhagic enteritis in order to establish the existence and prevalence of CPV-2c in Uruguay. During the study, analysis of the samples was done using PCR, RFLP and DNA sequence of the VP2 gene. 24 out of 25 samples were characterised as CPV-2c and one (1) as CPV-2a. Based on these findings, the study reported that CPV-2c was actually the prevalent field strain of CPV circulating in Uruguay.

Touihri et al. (2009) used MGB probe tools, sequencing and phylogenetic analysis to characterize CPV-2 variants circulating in Tunisia. A total of 79 samples were involved in the study (between 2007 and 2008) and 2 sets of primers were used for the screening. After screening and typing, it was revealed that the subtypes of CPV-2 were presently circulating in Tunisia with predominance for CPV-2b (42%).

Tajpara et al. (2009) used polymerase chain reaction in their study on the incidence of CPV in diarrhoeic dogs and found that the prevalence of CPV was high in dogs below 6 month of age (30.30%) compared to dogs over one year of age (15,60%). The prevalence of CPV infection was also remarkably high in unvaccinated dogs (31.40%) compared to vaccinated dogs (13.33%).

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Decaro et al. (2010) conducted a study to evaluate the detection rate of the CPV variants by using a commercial in-house test (SNAP canine parvovirus antigen test). Some 201 samples (58 faecal samples and 143 rectal swabs) were involved. The study found that in order for the *SNAP test to detect the variant agent, the sample must contain a viral load of >109 DNA copies/mg of faeces. However, some samples with a very high viral load tested negative using the in-house assay. The study suggested a possibility of antibodies interference in the samples or degradation of the capsid antigen due to long-term storage. Moreover, this study reported that to emphasise the diagnosis of CPV, the in-clinic assay was done first, followed by the laboratory confirmation with PCR- based assays in suspicious cases. CPV-2c was detected by the SNAP parvo test.

Ohshima et al. (2010) isolated a minute virus of canines (MVC) from an elderly dog with severe gastroenteritis using PCR and VI. In their study, one dog (11 years) showed signs of vomiting and bloody diarrhoea and a negative result was found when faecal specimen was tested for suspected CPV-2 infection. Therefore the study reported that MVC was non-pathogenic in aged dogs are in agreement with Carmichael et al. (1994). Pratelli et al (1991) stated that the most characteristic feature of bocaviruses (where MVC belongs) appears to be pathogenic in very young animal with mild to severe pneumonitis and/or enteritis in new born puppies.

Calderón et al. (2011) used PCR amplification for the identification and characterisation of CPV-2 subtypes in Argentina between the years 2003 and 2010. Amplification of a 583 bp fragment in the VP2 gene was perform on 79 rectal swabs collected from dogs suspected to be infected with CPV. The study reported that CPV was detected with predominance in puppies aged 1-5 months. The PCR result showed that CPV-2c was the predominant variant based on the AA substitution Glu 426.

Deka et al. (2013) also found that the prevalence of CPV was higher in males (21.12%) than females (8.45%). CPV positive was detected in vaccinated dogs (9.52%) and unvaccinated (30.15%). Deepa and Saseendranath (2002) also reported that 13.64% of vaccinated dogs suffered from CPV.

Streck et al. (2013) developed a PCR method using TaqMan technology for the detection and quantification of CPV-2 and FPLV in the serum and faecal samples of dogs. During the study, samples were subjected to several passage of different tests like HA, IF (Immunofluorescence), C-PCR and RT-PCR. The results revealed only 12% of the samples were detected by IF, 32%

Identification and characterisation of viral bloody diarrhoea aetiology in puppies, presented to the Animal Health hospital, North-West University